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THE LOCALIZATION of Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE

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THE LOCALIZATION of Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES VINCENT T. MARCHESI and GEORGE E. PALADE From The Rockefeller University, New York 10021. Dr. Marchesi's present address is the National Cancer Institute, the National Institutes of Health, Bethesda, Maryland 0014 ABSTRACT The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO3)2 resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb2+ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of Pi on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis Pi is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase. INTRODUCTION An ATPase' activated by Mg2+ , Na+ , and K+ , (4-9). Physiological studies on red cell ghosts and hereafter referred to as Na-K-ATPase, is found isolated squid axons have provided evidence that associated with cell membranes (1-3) and cell this ATPase is either part of or functionally as- fractions isolated from a wide variety of tissues sociated with the mechanism of active transport of Na+ and K+ (10, 11). Similarities between the I The following abbreviations are used in this paper: active transport mechanism and this ATPase ATPase, adenosine triphosphatase; ATP, adenosine activity include requirement for ATP and Mg 2+, triphosphate; ADP, adenosine diphosphate; CTP, + activation by Na and K+, and specific inhibition cytidine triphosphate; GTP, guanosine triphosphate, of both by the cardiac glycoside, ouabain. UTP, uridine triphosphate; Pi, inorganic phosphate; In addition to this Na-K-ATPase, SDS, sodium dodecyl sulfate; EDTA, ethylene di- each of the aminetetraacetate; DEAE-cellulose, diethyl amino- membrane and microsome preparations men- ethyl cellulose; NADH, nicotinamide adenine di- tioned exhibits an ATPase activity which is de- 2+ nucleotide, reduced; Tris, tris(hydroxymethyl) pendent only on Mg and is not inhibited by aminomethane. ouabain. This second activity, hereafter referred 385 to as Mg-ATPase, is presumably not involved in ghost membranes upon their ATPase activity was cation transport, but its exact relationship to the studied. Na-K-ATPase is not fully understood: the two activities have been ascribed to two distinct en- MATERIALS AND METHODS zymes or, conversely, considered as representing two different functional states of a single enzyme. Preparationof Red Cell Ghosts Both activities are known to be associated with Blood was obtained by cardiac puncture from the red cell membrane but their location in this adult guinea pigs under CO 2 narcosis, and ghosts membrane remains unknown. Previous investi- were prepared according to the following procedure: gators have attempted to determine sites of ATP- ase activity on membranes by using the lead-salt whole blood centrifuged 2,500 precipitation method of Wachstein and Meisel rpm, 15 min, with 0.1 MNa 3 citrate (12). However, objections to these studies have added as anticoagulant (5 ml/40 been raised on the grounds that the methods used ml whole blood) could not demonstrate Na-K-ATPase since this Plasma and Packed red cells washed activity was either destroyed by aldehyde fixation buffy coat twice in 0.9% NaCI, and re- or inhibited by the heavy metal used as capture aspirated off covered by centrifugation as reagent (13). Other objections have pointed out above; lysed in 10 vol of that, under the conditions of current cytochemical 5 mM Tris-HCI, pH 7.0, 1 mM procedures, nonenzymatic hydrolysis of ATP by EDTA,2 (a) mixed 15-20 lead could occur (14). min; centrifuged 25,000 g, 30 min; The present studies were initiated in an attempt r 1 to answer the following questions: (a) can a cyto- supernatant red cells resuspended in same chemical ATPase procedure be devised free of the discarded medium, mixed, centrifuged objections mentioned? (b) can the Na-K-ATPase 25,000 g, 10 min; of red cell membranes be demonstrated, and if so, supernatant (b) red cells suspended in where is this activity localized on the membrane? discarded 0.05 M Tris-HCl, pH 7.0, (c) can the Mg-ATPase be differentiated from the 0.50 M NaCI, 1 mM EDTA; 2 Na-K-ATPase on the basis of difference in locali- centrifuged 25,000 g, 10 min; zation on the membrane? supernatant red cells (now faint pink in We have chosen red cell ghosts as an experi- discarded color) suspended in 5 mM mental system since they consist of a single Tris-HCI, pH 7.0, 1 mM membrane and can be prepared easily, and since EDTA;2 centrifuged 25,000 g, their ATPase activity remains stable for relatively 10 min; long periods. Although compared to other tissues supernatant ghosts suspended in same me- (13) red cells are not particularly active, their discarded dium, centrifuged 25,000 g, 10 ATPase activity is well characterized and their min, membrane represents a system in which the corre- lation between active transport and ATPase ac- supernatant ghosts (now milky white) sus- tivity is reasonably secure (1, 2, 10). discarded pended in small volume (-0.5 ml/l ml pellet) of For these studies, a standard red cell ghost 0.05 M Tris-HCl, pH 7.0. preparation was made from guinea pig blood and experiments were carried out so that the most (a) This and all other operations after the first favorable conditions could be determined for the hemolysis were carried out in the cold. It was neces- cytochemical demonstration of both Mg- and sary to centrifuge the initial hemolysate at 25,000 g Na-K-ATPases. The responses of the red cell for 30 min to insure the complete sedimentation of all ghost system to prefixation by aldehydes and to the ghosts from the dense and viscous hemoglobin-con- presence of heavy metals used as capture reagents taining medium. This high speed was used in sub- sequent steps to shorten the centrifugation times. were investigated in light of the reports described (b) Treatment of ghosts with this high salt wash above. In addition, in view of data available on the ATPases of other membrane systems (15, 16), the 2 A 0.1 M stock solution of Na 2-EDTA, neutralized effect of detergents and of the physical state of the to pH 7.0 with 2 M Tris, was used. 386 THE JOURNAL OF CELL BIOLOGY VOLUME 35, 1967 was an important step in rendering the ghosts free of assayed for Pi by a modified Fiske-Subbarow proce- most of the residual hemoglobin. dure. The assay system contained 0.2 ml acidified The protein content of the final ghost preparations sample, 0.1 ml 2.5% NH 4 molybdate, 0.1 ml 1% was determined by the Lowry procedure (17) with Elon (Eastman Kodak Co., Rochester, N. Y.) as bovine serum albumin as the standard. reducing agent, and water to 1.0-ml volume. The optical density at 660 mg was linear with Pi concen- Ultrasonic Treatment of Ghost Membranes tration over the range used. Heat-treated ghosts (80 °, Ghost preparations were sonicated with a Branson 5 min) were used as controls so that ATP blanks Ultrasonifier for varying periods while kept immersed could be determined. All assays were carried out in duplicate. in an ice bath. Disruption of the ghosts to apparent 2 3 2 completion was monitored by phase-contrast micros- 32p RELEASE FROM [ p]-ATP: When [ p]_ copy. ATP was used as substrate, acid-washed Norit "A" (Amend Co., New York) was added to the HC104- Materials treated reaction mixture, and aliquots of supernate 32 containing nonNorit adsorbable p were dried on ATP and other nucleotides were purchased from planchets and counted in a Nuclear-Chicago Corpo- Sigma Chemical Co. (St. Louis, Mo.). The disodium ration (Des Plaines, Ill.) gas flow counter. Parallel salt of ATP was dissolved in water and neutralized to chemical determinations showed that the 32p of the pH 6.8 with Tris. ATP concentrations in stock solu- supernate was in the form of Pi. tions were determined spectrophotometrically with the assumption of a molar absorbancy of 15.3 X 103 at Cytochemical Demonstration of 259 mu. All other materials used in incubation media were of analytical grade when available. A TPase Activity 3 2 P-labeled ATP was prepared enzymatically ac- The incubation media contained Tris-maleate cording to the method of Glynn and Chappell (18) buffer, pH 7.0, appropriate cations (added as and was modified as follows. Under appropriate chlorides), ATP or other substrates, and Pb(N03)2 32 conditions pi was incubated with ATP, 3-phos- at concentrations given in the legends of the figures; phoglyceric acid, 3-phosphoglyceric acid kinase, the reagents were pipetted in the above order. The glyceraldehyde 3-phosphate dehydrogenase (all pur- Pb(NOa)2 was added to the medium while the tube chased from Sigma Chemical Co.), and NADH. was shaken on a Vortex mixer to prevent precipitation.
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